HIV-1 enters its target cell through fusion of the viral and cellular membranes in a process promoted by the viral envelope glycoprotein (Env). Upon gp120 engagement with celular receptors, the trimeric Env spike undergoes a regulated sequence of conformational changes in gp41 that result in formation of a highly stable six-helix bundle structure comprising the two heptad-repeat (HR) regions in the ectodomain of gp41. Whereas this postfusion motif is well-defined, structural details of the metastable prefusion and intermediate forms of gp41 that define the fusion pathway are lacking. Importantly, these prefusogenic states are postulated to mediate the antiviral activity of the fusion inhibitor enfuvirtide and the known broadly neutralizing antibodies. By characterizing the fundamental biophysical and structural properties of three newly identified 1-helical subdomains in gp41, we aim to elucidate the molecular basis of the structural transitions in the fusion pathway and thereby guide rational, structure-based efforts to design new immunogens and inhibitors. Our overarching hypothesis is that weak intermolecular interactions of gp41 that maintain the metastable state of prefusion Env are essential to prime membrane fusion activity. Therefore the design of soluble trimeric recombinant Env (gp140) protein candidates for vaccine and structural studies mandates protein engineering to stabilize the pleomorphic inter-subunit interactions. Oligomeric association of Env clearly depends upon gp41 ectodomain sequences, but present knowledge of the gp41-gp41 interfaces involved is rudimentary. By characterizing these interactions in detail, we aim to engineer stabilized forms of the cleaved HIV-1 gp140 trimer for structural analysis and thus help design new HIV-1 immunogens capable of eliciting broadly neutralizing antibodies. We propose: (1) To investigate the role of a putative prefusion trimeric subdomain formed by the C-terminal HR 1 region of gp41 in Env structure and function. We wil dissect the structural determinants of this autonomously folded subdomain and characterize the effects of mutations on the fusion potential of Env. We will make a trimeric, stabilized form of gp140 by using the HR1 substitutions that stabilize specific interfacial helical interactions in gp41. (2) To define the role of specific interactions in the membrane-proximal external region of gp41 that contribute to the stability and specificity of the trimeric Env complex. We hypothesize that trimerization of prefusion gp41 is promoted by formation of a labile trimeric coiled coil at the C terminus of the gp41 ectodomain. Results obtained in Aims 1 and 2 will be used to engineer a stable gp140 trimer for structural analysis. (3) To elucidate the structural properties of a novel 1-helical subdomain (called N26) of gp41 as a candidate molecular target for the entry inhibitor BMS-806. Our preliminary data indicate that BMS-806 interacts with the N26 subdomain formed by the N-terminal fusion peptide-proximal segment of gp41. To investigate the relevance of N26 to Env function, we will evaluate the effects of a series of rationally designed mutations on CD4-mediated activation of the membrane fusion cascade. PUBLIC HEALTH RELEVANCE: The research and public health communities concur that a preventive vaccine is the obvious long-term solution to bring the global HIV-1 epidemic under control. Unfortunately, this goal has proven elusive and no such vaccine is available. Overcoming this important biomedical problem will require further knowledge of structure-function relationships within the HIV-1 envelope glycoprotein complex.